Conditionally replicating viruses and methods for cancer virotherapy

Abstract

The present invention provides for methods and compositions for translation of a viral vector both in vitro and in vivo. Specifically, the present invention pertains to a translational control element placed in a vector to cause a selective translation of a viral vector. In one embodiment, the present invention provides for methods and compositions for conditionally expressing a viral vector inside tumor cells, while leaving normal cells unaffected due to their inability to translate the vector

1 . A conditionally replicating recombinant virus vector, comprising a replicating recombinant virus vector genome, comprising: (a) a replicating recombinant virus vector genome nucleic acid transcription sequence; and (b) a control sequence operatively linked to the transcription sequence; wherein the transcription sequence, when transcribed, produces a messenger RNA sequence that comprises an open reading frame encoding at least one viral protein necessary for replication, and a 5′-untranslated region (5′-UTR) sequence; wherein the untranslated sequence inhibits translation of the viral protein sequence under conditions that exist within normal mammalian cells that do not overexpress eukaryotic initiation factor eIF4E; and wherein the untranslated sequence allows translation of the viral protein sequence under conditions that exist within mammalian cells that overexpress eukaryotic initiation factor eIF4E relative to normal cells.  
   
   
       2 . The composition of  claim 1  wherein the untranslated sequence further comprises a secondary structure conformation having a stability measured as folded state free energy of ΔG≦ about −50 Kcal/Mol.  
   
   
       3 . The composition of  claim 1  wherein control sequence is a promoter.  
   
   
       4 . The composition of  claim 3  wherein the promoter is a tissue-specific promoter.  
   
   
       5 . The composition of  claim 3  wherein the promoter is an inducible promoter.  
   
   
       6 . The composition of  claim 1  wherein the virus vector genome comprises an adenovirus.  
   
   
       7 . The composition of  claim 1  wherein the virus vector genome comprises a type 2 or type 5 virus vector.  
   
   
       8 . The composition of  claim 1  wherein the virus vector genome is a type 2 or type 5 virus vector (Ad5) comprising an E1A gene operatively linked to the 5′-UTR sequence.  
   
   
       9 . The composition of  claim 2  wherein the 5′-UTR sequence is an oligonucteotide comprising at least a self-complementary sequence.  
   
   
       10 . The composition of  claim 9  wherein the 5′-UTR sequence comprises at least 30 nucleotides.  
   
   
       11 . The composition of  claim 10  wherein the 5′-UTR sequence is derived from sequences on genes selected from the group consisting of Fibroblast Growth Factor 2 (FGF2), proto-oncogene c-myc, cyclinD1, ornithine decarboxylase, and vascular endothelial growth factor (“VEGF”) .  
   
   
       12 . The composition of  claim 10  wherein the 5′-UTR sequence is derived from a Fibroblast Growth Factor 2 (FGF2) coding sequence.  
   
   
       13 . The composition of  claim 8  wherein the 5′-UTR sequence is upstream of the E1A mRNA coding sequence.  
   
   
       14 . The composition of  claim 1  wherein the viral genome codes for an oncolytic virus.  
   
   
       15 . The composition of  claim 1  wherein the virus is selected from the group consisting of adenovirus, HSV, vaccinia virus and parapoxvirus orf virus.  
   
   
       16 . The composition of  claim 1  wherein the virus is a recombinant virus from two or more types of viruses with differing pathogenic phenotypes such that it contains different antigenic determinants.  
   
   
       17 . The composition of  claim 1  wherein the viral genome is a recombinant adenovirus vector genome.  
   
   
       18 . The composition of  claim 17  wherein the recombinant adenovirus vector genome is encapsidated within an adenovirus capsid.  
   
   
       19 . The composition of  claim 18  wherein the adenovirus vector genome comprises AAV cap sequences.  
   
   
       20 . The composition of  claim 19  wherein the vector genome comprises from about 2.8 kb to 38 kb.  
   
   
       21 . The composition of  claim 17  wherein the virus vector genome comprises an early gene coding region operatively associated with a promoter selected from the group consisting of liver-specific, skeletal muscle-specific, cardiac muscle-specific, smooth muscle-specific, diaphragm muscle-specific, prostate-specific, and brain-specific promoters.  
   
   
       21 . The composition of  claim 17  wherein the virus vector genome comprises an early gene coding region operatively associated with a cancer cell specific promoter.  
   
   
       22 . The composition of  claim 1  wherein the virus vector genome comprises an adenovirus early gene coding region selected from the group consisting of E1, E2, E3, and E4 operatively associated with a cancer cell specific promoter.  
   
   
       23 . The composition of  claim 1  wherein the control sequence is a tissue-specific promoter derived from genes encoding at least one protein selected from the group consting of the prostate specific antigen (PSA), Carcinoembryonic antigen (CEA), secretory leukoprotease inhibitor (SLPI), alpha-fetoprotein (AFP), vascular endothelial growth factor, CXCR4 and survivin.  
   
   
       24 . The composition of  claim 23  wherein the tissue-specific promoter is an inducible promoter.  
   
   
       25 . The composition of  claim 23  wherein the tissue-specific promoter is the CXCR4 promoter.  
   
   
       26 . The composition of  claim 1  wherein the virus vector genome comprises an E1 coding region operatively associated with an inducible promoter.  
   
   
       27 . The composition of  claim 1  wherein the vector, when administered to a cell that overexpresses eIF4E, is effective to inhibit cell growth or lyse the cell.  
   
   
       28 . The composition of  claim 1  wherein the untranslated sequence allows translation of the viral sequence under conditions that exist within mammalian cells that overexpress eukaryotic initiation factor eIF4E at least 2-fold greater relative to normal cells.  
   
   
       29 . The composition of  claim 28  wherein the cell is a metastatic tumor cell.  
   
   
       30 . The composition of  claim 28  wherein the cell is a solid tumor cell.  
   
   
       31 . The composition of  claim 29  wherein the metastatic tumor cellis associated with a mammalian cancer selected from the group consisting of bladder, breast, cervical, colon, lung, prostate, and head and neck.  
   
   
       32 . The composition of  claim 1  wherein the virus vector genome further comprises a pharmaceutically acceptable carrier.  
   
   
       32 . The composition of  claim 1  wherein the virus vector genome further comprises a pharmaceutically acceptable carrier.  
   
   
       33 . A cultured cell comprising the conditionally replicating recombinant virus vector of  claim 1 .  
   
   
       34 . A pharmaceutical composition comprising the conditionally replicating recombinant virus vector of  claim 1  and a pharmaceutically acceptable excipient.  
   
   
       35 . A pharmaceutical composition comprising the conditionally replicating recombinant virus vector of  claim 1 , a chemotherapeutic agent and a pharmaceutically acceptable excipient.  
   
   
       36 . A method for conditionally expressing a replicating recombinant virus vector in a cell, comprising administering to the cell a DNA sequence comprising a replicating recombinant virus vector genome, comprising: (a) a replicating recombinant virus vector genome nucleic acid transcription sequence; and (b) a control sequence operatively linked to the transcription sequence; wherein the transcription sequence, when transcribed, produces a messenger RNA sequence that comprises an open reading frame encoding at least one viral protein necessary for replication, and a 5′-untranslated region (5′-UTR) sequence; wherein the untranslated sequence inhibits translation of the viral protein sequence under conditions that exist within normal mammalian cells that do not overexpress eukaryotic initiation factor eIF4E; and wherein the untranslated sequence allows translation of the viral protein sequence under conditions that exist within mammalian cells that overexpress eukaryotic initiation factor eIF4E relative to normal cells.  
   
   
       37 . The method of  claim 36 , wherein the untranslated sequence further comprises a hairpin secondary structure conformation having a stability measured as folded state free energy of ΔG≦ about −50 Kcal/Mol.  
   
   
       38 . The method of  claim 36 , wherein the untranslated sequence allows translation of the toxin sequence under conditions that exist within mammalian cells that overexpress eukaryotic initiation factor eIF4E at least 2-fold greater relative to normal cells.  
   
   
       39 . The method of  claim 36  wherein the administering is in an amount effective to inhibit cell growth.  
   
   
       40 . The method of  claim 36 , wherein the expression vector is delivered within a liposomal construct.  
   
   
       41 . A method for conditionally expressing a replicating recombinant virus vector in a cell, comprising administering to the cell an RNA sequence comprising a translatable messenger RNA sequence that comprises an open reading frame encoding at least one viral protein necessary for replication, and a 5′-untranslated region (5′-UTR) sequence; wherein the untranslated sequence inhibits translation of the viral protein sequence under conditions that exist within normal mammalian cells that do not overexpress eukaryotic initiation factor eIF4E; and wherein the untranslated sequence allows translation of the viral protein sequence under conditions that exist within mammalian cells that overexpress eukaryotic initiation factor eIF4E relative to normal cells.  
   
   
       42 . The method of  claim 41 , wherein the untranslated sequence further comprises a hairpin secondary structure conformation having a stability measured as folded state free energy of ΔG≦ about −50 Kcal/Mol.  
   
   
       43 . A method of treatment for a proliferation disease in a mammal, comprising administering to a mammal in need of such treatment a therapeutically effective amount of a DNA sequence comprising a replicating recombinant virus vector genome, comprising: (a) a replicating recombinant virus vector genome nucleic acid transcription sequence; and (b) a control sequence operatively linked to the transcription sequence; wherein the transcription sequence, when transcribed, produces a messenger RNA sequence that comprises an open reading frame encoding at least one viral protein necessary for replication, and a 5′-untranslated region (5′-UTR) sequence; wherein the untranslated sequence inhibits translation of the viral protein sequence under conditions that exist within normal mammalian cells that do not overexpress eukaryotic initiation factor eIF4E; and wherein the untranslated sequence allows translation of the viral protein sequence under conditions that exist within mammalian cells that overexpress eukaryotic initiation factor eIF4E relative to normal cells.  
   
   
       44 . The method as recited in  claim 43 , wherein the untranslated sequence further comprises a hairpin secondary structure conformation having a stability measured as folded state free energy of ΔG≦ about -50 Kcal/Mol.  
   
   
       45 . The method of  claim 44 , wherein the untranslated sequence allows translation of the sequence under conditions that exist within tumor cells that overexpress eukaryotic initiation factor eIF4E at least 2-fold greater relative to normal cells.  
   
   
       46 . The method of  claim 44 , wherein the untranslated sequence allows translation of the vial sequence within tumor cells in which the presence of eukaryotic initiation factor eIF4E allows the translation of the sequence, the viral genome is translated to produce viruses capable of substantial lyses the tumor cells.  
   
   
       47 . The method of  claim 44 , wherein the majority of non-tumor cells in the mammal are not killed due to the low levels of eukaryotic initiation factor eIF4E typically present in non-tumor cells  
   
   
       48 . The method of  claim 44 , wherein the encoded virus is a conditional virus.  
   
   
       49 . The method of  claim 44 , wherein the method additionally comprises administering an effective amount of a chemotherapeutic agent to the mammal.  
   
   
       50 . The method of  claim 44 , wherein the cancer is a metastatic tumor.  
   
   
       51 . The method of  claim 44 , wherein the cancer is a solid tumor.  
   
   
       52 . The method of  claim 50 , wherein the metastatic tumor is associated with a mammalian cancer selected from the group consisting of bladder, breast, cervical, colon, lung, prostate, and head and neck.  
   
   
       53 . A method of treatment for cancer in a mammal, comprising administering to a mammal in need of such treatment a therapeutically effective amount of a messenger RNA sequence that comprises a translatable sequence comprising an open reading frame encoding at least one viral protein necessary for replication, and a 5′-untranslated region (5′-UTR) sequence; wherein the untranslated sequence inhibits translation of the viral protein sequence under conditions that exist within normal mammalian cells that do not overexpress eukaryotic initiation factor eIF4E; and wherein the untranslated sequence allows translation of the viral protein sequence under conditions that exist within mammalian cells that overexpress eukaryotic initiation factor eIF4E relative to normal cells  
   
   
       54 . The method as recited in  claim 53 , wherein the untranslated sequence further comprises a hairpin secondary structure conformation having a stability measured as folded state free energy of ΔG≦ about −50 Kcal/Mol.  
   
   
       55 . The method of  claim 54  wherein control sequence is a promoter.  
   
   
       56 . The method of  claim 55  wherein the promoter is a tissue-specific promoter.  
   
   
       57 . The method of  claim 55  wherein the promoter is an inducible promoter.  
   
   
       58 . The method of  claim 55  wherein the virus vector genome comprises an adenovirus.  
   
   
       59 . The method of  claim 55  wherein the virus vector genome comprises a type 2 or type 5 virus vector.  
   
   
       60 . The method of  claim 55  wherein the virus vector genome is a type 2 or type 5 virus vector (Ad5) comprising an E1A gene operatively linked to the 5′-UTR sequence.  
   
   
       61 . The method of  claim 55  wherein the 5′-UTR sequence is upstream of the E1A mRNA coding sequence.  
   
   
       62 . The method of  claim 55  wherein the viral genome codes for an oncolytic virus.  
   
   
       63 . The method of  claim 55  wherein the virus is a recombinant virus from two or more types of viruses with differing pathogenic phenotypes such that it contains different antigenic determinants.  
   
   
       64 . The method of  claim 55  wherein the viral genome is a recombinant adenovirus vector genome.  
   
   
       65 . The method of  claim 55  wherein the recombinant adenovirus vector genome is encapsidated within an adenovirus capsid.  
   
   
       66 . The method of  claim 55  wherein the virus vector genome comprises an early gene coding region operatively associated with a promoter selected from the group consisting of liver-specific, skeletal muscle-specific, cardiac muscle-specific, smooth muscle-specific, diaphragm muscle-specific, prostate-specific, and brain-specific promoters.  
   
   
       67 . The method of  claim 55  wherein the virus vector genome comprises an early gene coding region operatively associated with a cancer cell specific promoter.  
   
   
       68 . The method of  claim 55  wherein the virus vector genome comprises an adenovirus early gene coding region selected from the group consisting of E1, E2, E3, and E4 operatively associated with a cancer cell specific promoter.  
   
   
       69 . The method of  claim 55  wherein the control sequence is a tissue-specific promoter derived from genes encoding at least one protein selected from the group consting of the prostate specific antigen (PSA), Carcinoembryonic antigen (CEA), secretory leukoprotease inhibitor (SLPI), alpha-fetoprotein (AFP), vascular endothelial growth factor, CXCR4 and survivin.  
   
   
       70 . The method of  claim 23  wherein the tissue-specific promoter is the CXCR4 promoter.